JPS6341426A - Carcinostatic agent - Google Patents

Abstract

PURPOSE:To prepare a carcinostatic agent that contains, as an active ingredient, a chemically modified protease originating from a microorganism. CONSTITUTION:The objective carcinostatic agent is prepared using, as an active ingredient, a chemically modified protease originating from a microorganism such as neutral protease. The protease is obtained by culturing a microorganism such as Serratia marcescence, Bacillus subtilis or Streptomyces griseus in an appropriate culture medium, collecting and purifying the enzyme from the culture mixture. The protease is chemically modified by coupling with a saccharide, introduction of hydrophobic polymer group, conversion of electric charge on the protein surface, binding with low-molecular-weight carcinostatic agent, formation of dimer by bonding between protease molecules, bonding with synthetic polymer polycation or bonding with synthetic polyanion. The dose is preferably 1-5,000mg/day, although a large dose can be given.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to an anticancer agent containing a chemically modified protease derived from a microorganism as an active ingredient.

Based on many years of experience in researching anticancer drugs, the present inventors conducted research to find an effective anticancer drug with fewer side effects, and first discovered that conventional anticancer drugs are completely different from each other in terms of molecular properties and mechanism of action. We have discovered that protease derived from microorganisms, which is a simple substance, has extremely strong anticancer effects.

Further research has led to the discovery in the present invention that an anticancer agent containing a chemically modified protease derived from a microorganism as an active ingredient has an even more excellent anticancer effect.

The action of chemically modified microbial-derived protease, which is the active ingredient of the anticancer drug of the present invention, is to degrade proteins and destroy cancer cells as a result, and has a simple action that is completely different from that of conventional anticancer drugs. It is an anti-cancer drug.

The present invention uses chemical modification to prevent the development of antibodies and, depending on the modifying substance, to enhance the destructive effect on cancer cells.

[Prior art]

The present inventors have previously demonstrated that in tumor tissues.

It was discovered that the structure of blood vessels and lymph vessels is significantly different from that of normal tissues. In other words, when a polymeric anticancer drug is administered locally to a tumor tissue, its behavior is completely different from that of a low-molecular substance; the polymeric substance is neither recovered nor excreted via the bloodstream or the lymphatic system, and remains active for a long period of time. They also discovered a phenomenon in which the tumor stagnates locally.

This is because the large molecular weight reduces the rate of spread of the drug and also reduces its passage through the vascular endothelium.
It is thought that this reduces the penetration into blood vessels and the subsequent recovery efficiency.

Furthermore, the present inventors have already discovered that lymph vessels are absent in the local area of solid tumors.

In normal tissues, the lymphatic system is the main route for recovery of macromolecules and oil droplets in these tissues, but because it is lacking in tumor tissues, macromolecules such as enzymes are It remains in the INWJ place for a long time.
Iwai, K. et al. Cancer Research (C
Ancer Res, ) Vol. 44, pp. 2115-2121, 1984, Maeda, Ecchi (Maeda, ) I
) et al. Journal on Protein Chemistry (J, Prot.

Chem,) Vol. 3, pp. 181-193, 1984, Hiroshi Maeda, Toshimitsu Konno, Cancer and Chemotherapy, Vol. 12, pp. 773-78.
2 pages 1985, and Maeda Ecchi (Maeda,
tl) et al. ``Protein Tailoring for Food and Medical Uses (r'rotein Tailo
ring for food and medical
tlses) pp. 353-382, edited by R9 Sleep Finney and J, R Wrightaker, Marcel Decka (Marca
ldekker) Published in 1986. ]] The present inventors focused on these findings, and when locally injecting microbial-derived protease, a polymeric substance,
Some of them exert strong and long-term effects on cancer cells, leading to the conclusion that they may have significant anticancer effects. [Problems to be solved] Anticancer drugs are mainly low-molecular-weight drugs, and even though they have excellent efficacy, they have the disadvantage that they tend to diffuse, making it difficult to maintain their efficacy.

[Means for solving problems]

Therefore, the present inventors conducted extensive studies to find a drug that has excellent anticancer effects, has long-lasting efficacy, and is low in toxicity to normal cells.

We also discovered that several types of proteases derived from microorganisms have significant anticancer effects on all experimental tumors in mice. It has been shown in vitro that the administered protease maintains its action for a long time in the cancerous local area, and its cytotoxicity to normal cells is considerably lower than that to cancer cells.
This discovery was made clear through an in vitro test, and a patent application (Japanese Patent Application No. 60-201607) was filed based on this knowledge as an anticancer drug containing a microorganism-derived protease as an active ingredient.

As a result of further research, the present inventors discovered that antibodies can appear in proteases, and their effects can neutralize the effects of proteases. The present invention was completed based on the discovery that the anticancer effect can be further enhanced by modification.

That is, the present invention relates to an anticancer agent containing a chemically modified protease derived from a microorganism as an active ingredient.

In preparing the chemically modified microorganism-derived protease that is the active ingredient of the anticancer agent of the present invention, the microorganism is first cultured, and the protease is collected from the culture.

Culture microorganisms and extract protease? :) Seratia marcescens (SeraLia marce)
Scens), Bacillus subtilis
s 5ubtilis), Strep 1-"
Streptomyces griseu
s), Bacillus sp.
), Streptomyces sp.
yces sp,) etc. are cultured in an appropriate medium,
It is obtained by collecting protease from the culture and purifying it.

Next, the proteases derived from these microorganisms are chemically modified. In this case, proteases produced by any of the above microorganisms can be similarly used, but in the present invention, in particular, Serratia marcetuscens (SerBLI) is used.
B marcescens) and Bacillus sp.
Bacillus sp.) protease is preferred.

Protease derived from Serratia marcescens (5 below)
The method for preparing Bacillus spp. 6 is shown in Preparation Example 1 below.
A method for preparing a protease derived from AT protease (hereinafter referred to as AT protease) is shown in Preparation Example 2.

Next, as a method for chemically modifying protease, there are various known methods [for example, J., Marshall et al.
Marshall etal, Journal of Biol Chem, Vol. 251, pp. 1081-1087, 1976. ) is used.

Namely, l) coupling with sugars, 2) introduction of hydrophobic polymer groups, 3) conversion of protein surface charges, 4) binding with low-molecular anticancer drugs, 5) dimer formation by binding between protease molecules. , 6) Coupling with a synthetic polymeric polycation, and 7) Coupling with a synthetic polyanion.

The chemically modified protease produced in this manner is administered into liquids, tablets, injections, suppositories, and the like.

Chemically modified proteases can be administered in large quantities, but administration of IB ~ 5000 mg/day is preferred.
N.

Next, preparation examples, production examples, test examples, and examples of the present invention will be described.

Preparation Example 1 Serratia, Marcescens ums 3958 FER
M-PN.

8436 was cultured overnight in 1-Lyptosoy liquid medium, and the 2°C MQ was added to IQ containing 200-350 mfl of the same medium.
Place in a Nosaka flask and shake (1-2Hz) at 30°C.
culture to reach the maximum equilibrium value of protease activity 2
After 5 to 30 hours, the culture solution is separated into a filtrate and bacterial cells, and the resulting Saitama filtrate is concentrated 4 times under reduced pressure at 30°C or below, or 90% saturated ammonium sulfate is added to form a precipitate, and then added to distilled water. On the other hand, dialysis (4°C, 48-72 hours) L, D
The protease was purified by adsorption of the active fraction onto an EAE-cellulose column, then elution under a gradient of NaCl, further dialysis, lyophilization, and then Sephadex G-100® treatment. The protease thus obtained was prepared in the presence of 001% Na dodecyl sulfate.

It was single by electrophoresis in a 7.5% polyacrylamide gel. The protease obtained here was named 56 protease. The enzymatic chemical properties of proteases are described in 56 by the present inventors in Journal on Bacteriology (J, Bacteriol), No. 1.
57 (1), pages 225-232, 1984.

Preparation agent 2 2 soluble starch, 1.5% soybean flour, 0.4 peptone
%, u mother X'f 0.2%, KH2PO40.1%,
and HPO40.3%.

MgSO4・7H, 00,01%, CaCl, -28,
12% medium containing 0.00,02% and CaC0°1.0%.
Sterilized by heating for 1'Cl2O, Bacillus sp.
RM-P Nα4736) was inoculated and cultured at 45°C for 24 hours. After sterilization by centrifugation, crude enzyme powder was obtained by precipitation with alcohol. The crude enzyme powder is dissolved in 2011M Tris-HCl buffer of pt+a,o containing 2mM calcium acetate and 1M ammonium sulfate, and applied to a Toyopearl 11υ55 (Toyo Soder, trademark) column equilibrated with the same buffer in advance to adsorb protease. . After washing with p++a,o 20mM I-Lis-HCl buffer containing 2+aM calcium acetate and 0.8M ammonium sulfate, pHl11. 20 of o
Purified preparations are obtained by elution with mM Tris-HCl buffer, dialysis against 20M calcium acetate, and lyophilization. This preparation showed a single band by SOS polyacrylamide electrophoresis using an acrylamide 12.5 gel.

The protease obtained here was named AT protease.

(Example of chemical modification of protease derived from microorganisms) Production example 1 Dextran (manufactured by Pharmacia, average molecular weight approx.
000) and dissolve it in 9 ml (l) of purified water.

To this, 1 mQ of 10% sodium metaperiodate was added, and the mixture was allowed to stand overnight in a cool, dark place at 4°C for activation. Then, 30% sodium bisulfite was added little by little to reduce excess iodine. After the color of iodine disappears, salts are removed by thorough dialysis against purified water, and activated dextran is obtained by freeze-drying.

Next, take 20 mg of activated dextran powder, add 10 mg of purified AT protease, and 10 mg of O, 1M boric acid a solution.
Dissolve in m11 and react overnight. Sephacryl S-3
Unreacted substances were separated by gel filtration using a 00 (Pharmacia Co., Ltd.) column, followed by dialysis and freeze-drying to obtain a final sample.

When protease activity was measured by casein decomposition ability, the activity yield was 63%.

Production Example 2 Dextran sulfate (manufactured by Sigma, molecular weight approximately s, ooo)
Take 100mg and add 0.4N Na01147m12
Add 3tsQ of epichlorohydrin, and react at 40°C for 2 hours with stirring. The reaction solution was neutralized with IN IIcI and then evaporated to dryness using an evaporator. Next, it is dissolved together with AT protease 110l11 in 0.1M borate buffer (pl+9.0) containing 2IIIM calcium acetate, and reacted overnight at 4°C. The reaction product is filtered through a membrane filter with a pore size cutoff of molecular weight -10,000 to remove unreacted dextran sulfate. When the reaction product was subjected to SDS polyacrylamide electrophoresis to check the molecular weight, it was found that AT protease (molecular weight 37,50
0) disappears, and a larger range (40,
A band was detected between 000 and 45,000). The product was dialyzed against 2MM Cu21'' and then lyophilized.

Production example 3 20g monomethoxypolyethylene glycol at 100＠
Activated polyethylene glycol (polyethylene glycol is abbreviated as PIEG) was obtained by dissolving it in benzene and adding 365ml of cyanuric chloride, reacting at 80°C for 44 hours, and precipitating with petroleum ether.

Next, 30a+g of AT protease was dissolved in 15d of 0.1M borate buffer (pH 8), and activated PEG 500
mg was added and reacted at 37°C for 1 hour.

The reaction solution was subjected to gel filtration using a Sephacryl S-200 column to collect both components with a molecular weight of 50,000 to 100,000, which were dialyzed and freeze-dried to obtain 5 mg of a sample. Protease activity yield was approximately 10%.

Production example 4 30mg of 56 and 2mfl of protease 0.8M
Dissolved in Na1lC03, added 10 mg of styrene maleic anhydride copolymer (hereinafter abbreviated as SMA), and heated at 4°C.
Allow to react for 16 hours. The reaction solution was diluted with 0.1M borate buffer (
After dialysis against pl+8.5), Sephacryl S-
The product was subjected to gel filtration using a 200 column, and both fractions each having a molecular weight of approximately 60,000 were taken, followed by dialysis and freeze-drying to obtain a sample of 5B.

Production Example 5 20 mg of AT protease was dissolved in 10 mm of 0.01 M imidazole buffer, 25 mg of succinic anhydride was gradually added as a powder, and reacted for 2 hours with an old IN NaO trowel while keeping the pH neutral. was dialyzed against the same buffer, and then DEAE-cellulose equilibrated with the same buffer (
Howa Sotoman Co.) column to adsorb it, and then 0 to 0.5
Perform linear gradient end elution with MNaC1,
Both fractions eluted at a higher salt concentration (0.4 M) than the original protease were collected, dialyzed, and lyophilized. The entire surface of the obtained enzyme protein had a negative charge.

Production Example 6 Take 20 mg of protease in 56, dissolve it in 4tQ of 0.01M calcium acetate solution, and add 2M N, N
-Dimethylethylenediamine aqueous solution (pH 8) 5 t
Add afi.

Further, IM water-soluble carbodiimide solution LmQ was added, and the mixture was reacted at 4°C overnight, then dialyzed against a 2mM calcium acetate solution, and freeze-dried. When subjected to 7.5% polyacrylamide electrophoresis (PH4,0), the original 56
The band shifted to the negative electrode side compared to the protease, confirming that it had salt-like properties.

Production example 7 Take 20+*g of AT protease and add 2 +*MCu”+
Dissolved in 10 mQ of Tris buffer containing 0.01 N
- Add 170 μΩ of a 5 mg/ml ethanol solution of succiimidyl-(pyridyldithio) propionate (hereinafter abbreviated as 5pop) little by little, react for 30 minutes at room temperature, and desalt using a Sephadex G-25 (manufactured by Pharmacia) column. did.

On the other hand, poly-lysine (Sigma Co., Ltd., molecular weight approximately 47,000
0) Take 13 mg and perform the same operation. Now, add dithiothreitol to the Aτ protease solution to a concentration of 0.1 M, immediately desalt it, and combine it with the AT protease having a thiol group to form a 5-pop poly-protease. By mixing lysine and reacting overnight at 4°C, poly-L-lysine and AT protease bonded via disulfide bonds can be obtained. Unreacted substances were separated using a Sephacryl S-300 column, followed by dialysis and lyophilization.

Production Example 8 Protease, the low molecular weight anticancer drug methotrexade (hereinafter abbreviated as MTX), and water-soluble carbodiimide were mixed in a molar ratio of 1:50:500 in 0.1 M Tris-HCl buffer (pH 8,0) for 10 s + Q. ℃ overnight reaction,
The mixture was then dialyzed against a 2mM calcium acetate aqueous solution to remove unreacted low molecules and salts, and freeze-dried. Uv spectrum analysis revealed that 4 MTX molecules were bound to 1 molecule of protease.

Production Example 9 DNA was used instead of the low molecular weight anticancer drug MTX in Production Example 8.
A reaction was carried out in the same manner as in Production Example 8 using cytosine arabinoside (hereinafter abbreviated as ara-c), which is a polymerase inhibitor, to obtain an AT protease cytosine arabinoside conjugate.

Production Example IO 0 in which proteases were formed to form intermolecular crosslinks by disulfide bonds in 56 using 5pop described in Production Example 7.
The reaction product was separated into duplexes and unreacted monomers using a Sephacryl S-200 column, and only the duplex portion was taken, dialyzed, and lyophilized.

Production Example 11 20 mg of protease was added to 56 in 0.05 M borate buffer (p) containing 2 mM calcium acetate +9)9. Omfl
and add 1n+fl acetone. While stirring, 40 mg of NaBIl was added little by little as a powder over 1 hour, gel filtrated through a Sephadex G-25 column to remove unreacted low molecules, and then freeze-dried. As a result, the amino group of the protease was replaced with an isopropyl group, increasing its hydrophobicity and lowering its isocenter (
The migration of isocenters was confirmed by 75% polyacrylamide gel electrophoresis. ).

Note that the method for measuring protease activity used in the present invention will be described below. Substrate milk casein (1,5% V
/V, 0.1M phosphate buffer pH 8,0) 1 m
(IL,; Add 1 sM enzyme solution (diluted with the same buffer), react for 30 minutes at 37°C, then add 2 mfl of 0.4 M trichloroacetic acid to stop the reaction, and centrifuge the coagulated denatured protein. The absorbance of the supernatant at A*at+ll- is measured.

Test Example I Crj: BPF, 8-1 subcutaneously in the flank of strain mice
6 melanoma cells were inoculated at 2.5 x 10” per animal, and
o-12 days later, the tumor volume had grown to approximately 200 s + sm'', and each chemically modified protease prepared at a predetermined concentration was administered in a single dose. lligh
dose: 3゜3mg/ma), moderate dose (
Middle dosa: 1.011 ward/-phantom, low dose (Low dose: 0.33+mg/mf
There were three dosage levels for i). In addition, the protease solution at each concentration was adjusted to 0.11 Ω per tumor in accordance with the size of solid tumors in mice. Protease lysis is 5
6, protease and its modified form in physiological saline, A
For T protease and its modified forms, physiological saline containing 2mM calcium acetate was used.

The administration port was set as day 0, and thereafter on the 1st, 4th, 7th day, lO
Day 0 tumor size was measured. The size is measured by measuring the major axis, minor axis, and height using a caliper (unit: am) (long axis/2x [diameter/
2) Calculated as XKX height=volume unit Iam'''.

The results are shown in Table 1. The effect was determined by measuring the rate of increase in tumor volume due to each modified protease when the tumor size on the 4th and 7th day was φ, and the rate of increase in tumor volume when administering unmodified protease was 1. It was shown by The smaller this value is, the more the antitumor activity of the modified protease is increased compared to the original protease.

Table 1 Increase rate of tumor volume 56K to 1.0056-
PEG O, 8056 to 5N
A 0.7156 monosuccinic acid 0.7256 monomethotrexate 0.6356 monodimethylethylenediamine 0.8456 monoalkylated 0.
8256-double body 0.92AT
1.00AT-dextran sulfate 0.74AT-PEG
O, 31AT-5MA
O,5gAT-succinic acid 0.63AT-dimethylethylenediamine 0.82AT-methotrexate 0.5S AT-cytosine arabinoside 0.71AT-dimer 0,76AT-dextran 0.53 control
3.80 56 in Table 1 is an abbreviation for protease, and AT is an abbreviation for AT protease. Also, the control is the case without protease.

As is clear from Table 1, the chemically modified protease derived from microorganisms has a stronger antitumor effect than the original protease.

Test Example 2 Acute toxicity test of various chemically modified proteases
A group of 10 mice each weighing 25 g of dd' were given intravenous or intraperitoneal administration of various chemically modified proteases dissolved in physiological saline or physiological saline containing 2IIIM calcium acetate, and the symptoms were maintained for one week. No abnormality was observed in any cases of protease administration.

As is clear from the above, chemically modified proteases derived from microorganisms have been found to have significant anticancer effects.

Therefore, the practical application of anticancer agents containing proteases derived from these chemically modified microorganisms as active ingredients is highly anticipated.

When these chemically modified proteases derived from microorganisms are administered to humans, they can be effectively treated by direct intratumoral, intraperitoneal, or oral administration.

The dosage of these chemically modified proteases derived from microorganisms is not constant depending on the size of the tumor, growth rate, etc., but it is usually 1 mg to 10 μg into the tumor.
~Inject in several places. In addition, for the peritoneal cavity, other disseminated intraperitoneal tumors, or ascites cancer, 1 to 200 ml of a diluted chemically modified microbial protease solution is usually used.
Inject Q intraperitoneally.

Furthermore, in the case of oral administration, 1 mg to several g per adult is usually administered at once or in divided doses.

The dosage form can be powder, aqueous solution, granule, capsule, enteric-coated, or oil-solubilized. Also, as a mixture, gastric fluid can be used to inactivate these chemically modified proteases derived from microorganisms. It is also possible to use pepsin inhibitors to protect against pepsin in the drug.

Examples of the present invention are shown below.

Example 1 PEG-modified AT protease 100 produced in Production Example 3
mg is dissolved in 1On+Q physiological saline containing 2mM calcium acetate, filtered aseptically using a membrane filter, the filtrate is filled into a sterilized glass flask, freeze-dried, and this is tightly stoppered to freeze for injection. It was made into a dry powder.

Example 2 100 g of PEG-modified AT protease powder obtained according to Example 1, 97 g of milk &'f and 3 g of magnesium stearate were each weighed and mixed uniformly, and this was placed in Nα2 gelatin capsules. 200
After filling each mg in mg, an enteric coating was applied to prepare enteric-coated capsules.

Example 3 200 mg of the dextran-bound AT protease produced in Production Example 1 was dissolved in 1011IQ physiological saline, filtered aseptically using a membrane filter, and the filtrate was filled in sterilized glass containers with lomR portions and freeze-dried. This was sealed to obtain a freeze-dried powder for injection.

Example 4 1 g of dextran-bound AT protease powder obtained according to Example 3 and 84.5 g of crystalline cellulose.

After weighing 1.5 g of mannitol log, carboxymethylcellulose calcium, and hydrogenated oil, the uniformly mixed powder was granulated using an extruder to produce granules for internal use.

Example 5 500 mg of protease was dissolved in 10 mQ of physiological saline to the methotrexate bond 56 produced in Production Example 8,
Filter aseptically using a membrane filter.

The filtrate was filled into a sterilized glass tube at a volume of 10 mR each and freeze-dried, and the tube was tightly stoppered to obtain a freeze-dried powder for injection.

Example 6 To the methochrexate bond 56 obtained according to Example 5, 1 g of protease powder, 84.5 g of crystalline cellulose,
Milk & HOg, carboxymethyl cellulose calcium 2
g, magnesium stearate 1g, stearic acid 1.
5g of powder was mixed well and uniformly, and then it was made into a tablet with a weight of 10g by a tablet machine.
After producing a 0 mg plain tablet, this tablet was coated with an enteric coating agent to produce an enteric coated tablet.

[Effects of the present invention]

The anticancer agent of the present invention, which contains a chemically modified microbial-derived protease as an active ingredient, has the features of sustained drug efficacy and low toxicity to normal cells. Furthermore, possible immunological reactions upon frequent or repeated administration can be avoided.

Claims (1)

[Scope of Claims] 1. An anticancer agent containing a chemically modified protease derived from a microorganism as an active ingredient. 2. The anticancer agent according to claim 1, wherein the microorganism-derived protease is a neutral protease.